Biogeochemical analysis of soil samples from Robinson Ridge

Soil samples were collected from Robinson Ridge in the 2021-22 field season and subject to metagenomic and physicochemical analysis.General site description Outcrops to 87m covered in small cobble-sized gravel with small boulders interspersed through these gravel fields. The North face of the larger Eastern outcrop showed steep exposed bedrock above an extensive talus field (cliff debris). Lower elevation (including talus field), and drainage lines, small gullies and meltwater channels on all faces held moss beds or dense lichen communities. At higher elevations moss was largely absent, although gravel fields still contained lichen (ubiquitous distribution) and sub-lithic algal communities (patchy distribution). Lichen occurred on all rocks down to cobble and pebble sized gravel at the surface, irrespective of aspect or position in relation to water sources. Although lichen varied in density it was ubiquitous across all exposed rock sites.Specific site descriptionAt higher elevations small drainage lines ran among drier soil hummocks. Lichen coverage was noticeably denser in drainage lines. Lichen coverage on dry soil hummocks was sparse and restricted to larger gravel rocks. Once these gravel rocks were removed from the surface no lifeforms were visible in the dry topsoil. This was targeted for sampling. A distance greater than 100m to moss, animal activity and obvious algae cover was possible at all sites; however, maintaining a distance greater than 100m to lichen and small sub-lithic algal communities was not possible due to the ubiquity and low detectability, respectively. Sampling procedureAll sampling gear was cleaned with ethanol in the lab prior to taking into the field and cleaned with ethanol between sites if used more than once. Hair cover (beanie), face mask and nitrile gloves were worn during sampling.To remove lichen from samples, top gravel was removed from a sample location prior to sampling. If sub-lithic algal communities were observed whilst removing gravel from a sample location, then a new sample location at the same site was chosen. Triplicate ~500 g top soil (0-10 cm depth) samples were collected at each of the three sites. Soil samples were transported and stored frozen at -18˚C until analysis commenced in 2024. SamplesRR1 66.367046°S 110.594036°E Near top of a knoll on Eastern outcrop of Robinson Ridge. North facing aspect with slight slope. Patchy snow cover surrounding site. Triplicates taken within a 1.5m radius.RR2 66.367506°S 110.591279°E Flat top of a knoll on Eastern outcrop of Robinson Ridge with very slight South facing aspect. Relatively more lichen in area than Site RR1. Deep snow patches nearby. Soils taken within a 1.2m radius.RR3 66.369639°S 110.583777°E Gentle North-West facing slope close to top of highest knoll on Western outcrop of Robinson Ridge. Some snow patches. Lichen coverage more than Site RR2. Soils taken within a 4m radius.DNA extraction and metagenomic analysisDNA was extracted from two pooled 0.5 g soil samples from each site (n=3) using the FastDNA SPIN Kit for Soil (MP Biomedical) with a modified protocol for low biomass extraction, in which the technical duplicates were pooled during the binding matrix step, then washed and eluted as one. DNA was quantified using the Qubit HS dsDNA assay. The extracted DNA was submitted to the Genomics Node of the Monash Genomics and Bioinformatics Platform (Melbourne, Victoria) for library preparation and sequencing, which was carried out on a single lane of a NovaSeqX Plus (Illumina) using XLEAP-SBS chemistry (2 × 150 bp). Full metagenomic assembly and analysis details are in a forthcoming paper, which will be linked upon publication. Metagenomic reads are available on the NCBI Sequence Read Archive under PRJNA1272669 and MAGs are available on NCBI under BioProject PRJNA1272656 and PRJNA1272657.Soil physicochemical analysisSoil gravimetric soil moisture content was determined by calculating the weight difference (to 4 decimal places) of 5 g soil after drying at 70˚C for one week, followed by 105˚C for seven hours. Samples were placed in a desiccator to return to room temperature before weighing. All remaining physicochemical analyses were performed by Environmental Analysis Laboratory at Southern Cross University, after soil samples (50 g) had been irradiated with 50 kGy gamma irradiation to comply with Australian Department of Agriculture, Forestry and Fisheries biosecurity requirements for quarantine samples. Analyses performed included pH, electrical conductivity, Effective Cation Exchange Capacity (ECEC), elemental analysis (P, NO3-, NH4+, S, Ca, Mg, K, Na, Al, H, Mn, Fe, Cu, B, Si, Ca:Mg ratio, total N, total C), total organic carbon and total organic matter. Results are in the associated data record, but embargoes until publication. Trace gas oxidation rate assaysTo ascertain rates of hydrogen (H2), carbon monoxide (CO) and methane (CH4) oxidation and the thermal sensitivity of these processes, soils were incubated at 14 different temperatures (-20, -8, 4, 10, 17, 25, 30, 37, 42, 50, 60, 70, 75 and 80°C). One pooled, heat-killed sample was included as a negative control at each temperature. Soil (1-10 g) was aliquoted into 120 ml glass serum vials, and then left to equilibrate for two hours at the incubation temperature, before being sealed with NaOH-rinsed butyl rubber stoppers and aluminium crimp caps and adding 10 ppm each of H2, CO, and CH4 (procured as a premixed gas in N2) was added to an ambient air headspace. At each time point, 2 ml gas samples were withdrawn via a syringe fitted with a stopcock and stored in 3 ml glass exetainers sealed with silicone as described previously [https://doi.org/10.5194/bg-18-729-2021]. Gas sample exetainers were analysed on a VICI gas chromatograph with a pulse discharge helium ionisation detector as previously described [https://doi.org/10.1038/s41396-019-0393-0] and quantified by comparison to a three-point standard curve (0, 10, 100 ppm each of H2, CO, CH4). Trace gas oxidation rates were determined by fitting exponential growth curves to the depleting gas concentrations over time, normalised to controls. Trace gas oxidation rates will be provided in the Supplementary Information of an upcoming paper, to be linked upon publication.